GENERAL PRACTICAL (I) INTRODUCTORY LECTUREipmbgazette.weebly.com/uploads/1/0/3/0/1030249/practical_ppt_1.pdf · INTRODUCTION Objectives: General practical (I) preparatory lecture

GENERAL PRACTICAL (I) INTRODUCTORY

LECTURE

Steven Odongo [email protected] /[email protected]

CMIM, Building E, Floor 8, Room E8.07

14/11/2011 Steven Odongo, Dep't of Structural Biology, CMIM

mailto:[email protected]

mailto:/[email protected]

INTRODUCTION

Objectives:

General practical (I) preparatory lecture.

Good laboratory practice (GLP) familiarization (see

manual)

Overview of basic laboratory techniques.


MAKING SOLUTION

(Reagents & Buffers)

a) Basic calculations

Molar solution

Percent solution

Concentrated stock solution

Dilution of stock solution


Molar solution

Molarity = No. of grams MWs (moles) solute/1 L solvent

Calculation of Molarity (M)

Know final volume (V), final concentration (M) & FMW of

solute.

E.g. Prepare 1M NaCl (FMW=58.43) in 1L water (H2O)?

Weigh 58.48g NaCl in a cylinder and fill with H2O to 1L (1000ml) mark


E.g. Prepare 2M NaCl (FMW=58.43) in 1L H2O?

Weigh (2x58.43)g NaCl and fill with H2O to 1L mark

Sometimes, to be quick, use formula:

E.g. Prepare 5M NaCl (FMW=58.43) in 0.5 L (500ml) H2O?

FMW (g) x final volume (L) x final concentration (M) = grams required

58.43 x 0.5 x 5 = 146g


Percent solution

Percent solutions = based on 100ml or 100g

Expressions of concentration in percent:

Percent weight by volume - % (w/v)

Percent volume by volume - % (v/v)

Percent weight by weight - % (w/w)


Percent weight by volume - % (w/v)

Grams of solute per 100 ml of solvent

E.g. 20% Glucose = 20g glucose dissolved in 80g (80ml) water.

Percent volume by volume - % (v/v)

ml of solute per 100 ml of solvent. E.g. 1% SDS solution = 1ml SDS dissolved in 99ml water.

Percent weight by weight - % (w/w)

Grams of solute per 100g of solvent.

E.g. 10% sucrose solution = 10 g sucrose dissolved in 90 g water.


Concentrated stock solution

Buffers may be prepared or provided as a concentrated stock (2x, 5X 10X etc…), and diluted to a working concentration (usually 1x)

E.g. a 10x phosphate buffered saline (PBS) per 1L of solution

requires 10 times every reagent used for preparation of 1L

1xPBS.

1xPBS = 8g NaCl, 0.2g KCl, 1.3g Na2HPO4.2H2O, 0.24g KH2PO4,

top with water to 1L.

For 10xPBS, multiply every reagent by 10.


Dilution of stock solution

Formula used commonly: Ci x Vi = Cf x Vf Ci = initial concentration, or concentration in stock solution.

Vi = initial volume or amount of stock solution needed.

Cf = final concentration or concentration in desired solution.

Vf = final volume of desired solution.

E.g. 1) From 1.5M NaOH, prepare 100 ml of 0.05M solution Ci = 1.5M, Vi = ?, Cf = 0.05M, Vf = 100 ml 0.05 x 100 = 1.5 x Vi

Vi = 3.33 ml of 1.5M NaOH (stock), add H2O to 100 ml

2) Prepare 100 ml 1 X solution of PBS buffer from a 10 X stock

solution



b) Weighing and Mixing reagents

wighing boats Spatulas

Cylinder

Beaker

Erlenmeyer flask

Magnetic stir plate Magnetic stir rod

Weighing balance

Weighing:

Put on nitril/latex gloves

Read instruction on the bottle (bse may require wearing mask)

Turn on the balance & tare the weigh boat

Weigh your material & pour it into the beaker or flask ( may

beaker or cover flask immediately)

Close the lid of the stock jar & put in the shelf immediately

Clean the balance when finished


Mixing:

Add 80% of the total final volume of water to the beaker

followed by solid.

Drop magnetic stir bar of an appropriate size

Put beaker on the magnetic stir plate & slowly turn on

the magnetic stir plate

Stir until complete dissolution occurs

Pour solution into the graduated cylinder bring volume to

90%

Pour solution back into the beaker for pH-ing, label with

tape



C) pH measurements and adjustments

Calibration of pH meter

Required: At least 2 pH standard solution (pH 4,7&10), 3 or

4 50ml beakers, wiping paper

Rinse electrode and gently dry on wiping paper

Pour ~1.5 inches of standard

Press “standardize” and wait until pH stabilizes to

standard’s (e.g pH 4)

Press “standby”, rinse electrode and dry

Repeat standardization for pH of 10 if not yet done.

Rinse electrode, put back in storage buffer

Read the pH of standardization buffers


Determining pH Requirements:

Magnetic stir bars and plates,

(12.1 & 1)M HCl,

(5 or 10)M NaOH & 0.1M NaOH.

Procedure:

Stir the the solution on stir plate. Stir bar should rotate very slowly to avoid damaging electrode.

Raise electrode out of soak beaker and rinse, then immerse into the solution which the pH is to be measured

Chang e the function switch from standby to pH

Wait for the pH to stabilise and adjust by adding NaOH or HCl


Add a drop of (NaOH or HCl) at a time, stir, then measure

If the pH is off by a unit, use more conc acid (12.1M HCl) or base (5 or 10M NaOH).

Don’t use the same pasteur pipette for acid and base addition

You will need more drops near pH at which the solution is buffered.


Transfer the solution into a measuring cylinder, bring the volume to 100%

Pour the solution into glass or plastic bottle with cap

Label bottle:- date, components, concentration, pH and your initials.

Put apiece of autoclave tape. Dark strips appear after autoclaving.

If not autoclaving immediately keep at 4°C


D) Sterilizing solution:

1) Autoclave:

Most buffers

Undefined bacterial and yeast media

But don’t autoclave:

Corrosives (acids, bases & phenols), solvents or volatiles (the Ohs, Chloroform), or radioactive materials, liquids containing beach (sodium hypochlorite), formalin, or glutaraldehyde, heat labile ingredients (serum, antibiotics, proteins & vit), mammalian, plant, and insect media, HEPES containing solution, DTT or BME

solutions, buffers with detergent such as 10%SDS.


2) Filtration

Heat labile or volatile or solution less than 20ml.

Viscous solution is pre-filtered through 0.4µm

pore. a 0.2 µm (most media and buffers) or 0.1µm

(tissue culture media)

Note that virus will pass through filter hence can

be removed by filtration



Syringe filters

Vacuum filters

e) Storage of buffers and solutions

Store media at 4°C

Buffers stored at room temp or 4°C

Concentrated solutions at room temperature

Light sensitive reagents store at appropriate temperature

and kept in a brown bottle or cover with foil or in a box.

f)Discard buffers

Whenever discoloration occurs

Contamination appears

Precipitate persists after warming in water bath at 37°C

for 20min (or check for salt cyrstals under microscope

X100). Salt crytals are large while contaminants are uniform and small


DNA TECHNIQUES

Plasmid DNA & Plasmid

isolation

DNA restriction digestion

Ligation (joining)

Transformation

Polymerase chain reaction

(PCR)

Agarose gel electrophoresis

DNA molecular weight

determination


a) Plasmid DNA

Circular molecules of

independent extragenomic DNA in

bacteria

Posses one or more vital genes

e.g. Antibiotic resistance

Most have atleast one ori-able to

multiply independently of the

bacterial x-some.

Some plasmids (episomes) insert

into the bacterial x-some for them

to replicate and at alter stage

excised to exist as independent

element.



Artificially modified Plasmid DNA

b) Isolation of plasmid DNA

Alkaline denaturation



c) Determining Nucleic Acid Concentration

& Purity by UV spectroscopy



D) DNA restriction digestion and ligation


E) Transformation

Transformation=introduction of foreign DNA into bacterial

cell (may also include plant, mammalian cells).

May be natural or artificial

Before bacteria are artificially transfromed they ahve to made

competent-ability to take up DNA

1) Chemical transformation


Alternatively, bacteria can acquire

foreign DNA through:

Conjugation ► Mating btn bacteria

Transfection ► Bacteriophages

inject the foreign DNA


2) Electroporation


F) Selection of transformants (Antibiotic resistance)

When bacteria are transformed in the

laboratory, it acquire new traits from the

transformation plasmid.

These traits are easily identifiable and

allow for selection of transformed cells.

E.g. the bacteria transformed in here

acquire resistance to the antibiotic

tetracyclin.

You prove this by growing them up on

LB:tet plates.

Untransformed bacteria will not grow on

the LB:AMP plates.

G) Transformation efficiency

Measure of the amount of cells within the bacterial culture able to take up DNA molecules.

Determined experimentally e.g. to detemine transformation

efficiency of an intact plasmid DNA at two different concentrations (1x & 10X):



H) Polymerase chain reaction (PCR)


I) Agarose gel electrophoresis to detect DNA

-

+

Before electrophoresis After electrophoresis



J) Ethidium bromide visualization of DNA bands

K) Determination of DNA molecular weight

Rf = Distance of DNA migration from the origin

Distance of migration of dye from the origin


Lo

g M

W

Relative mobility (Rf)

y=mx+c

PROTEIN TECHNIQUES

a) Expression of recombinant protein



E.g. Expression of Nanobody

•All of these are possible with a few simple genetic manipulations


b) Protein expression, extraction and purification


c) Protein detection

I) Sodium Dodecyl Sulphate polyacrylamide gel electrophoresis (SDS PAGE)

Principle


Components of SDS-PAGE and

mechanism of polymerization

Relative protein migration vs SDS-

PAGE concentration


SDS-PAGE Steps


II) Western blot to detect protein

Indirect detection

a) SDS PAGE

c) Immunoassay

b) Blotting

Which of these bands is my protein of

interest? Is there Ab

specific to my protein? Yes, then do WB

Log MW (kDa) against Rf values

y = -1.1015x + 2.0976

R2 = 0.9935

0

0.5

1

1.5

2

2.5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Rf values of Protein Standard

Lo

g M

W (

kD

a)

Protein

marker 1 2 3 4 5 6

Rf (X) 0.15 0.29 0.42 0.54 0.74 0.87

MW 92 60 40 30 20 14

Log MW

(Y) 1.96 1.78 1.6 1.48 1.3 1.15

(Rf) = Distance of DNA migration from the origin

Distance of migration of dye from the origin

Rf value of sample = 0.56, MW ?

Calculation

Y = -1.1015x + 2.0976

= -1.1015 (0.56) + 2.0976

= 1.48

Therefore, the molecular weight of the

sample equals to the antilog of 1.48 =

30.3kDa

d) Calculating MW of protein after SDS-PAGE


e) Determination of protein concentration (BCA)



From the stock solution of 2 mg/ml of BSA prepare 1.8 mg/ml, 1.6

mg/ml, 1.4 mg/ml, 1.2 mg/ml, 1.0 mg/ml, 0.8 mg/ml, 0.6 mg/ml, 0.4 mg/ml

and 0.2 mg/ml.

Prepare 2-fold serial dilutions of sample as indicated in the diagram

of microplate

Fill wells of a flat-bottom micro-titer plate by pipetting 10 µl of each

& blank well filled with PBS

Mixing reagents A and B in a 50:1 proportion

Incubate plate at 37°C for 30 mins or at RT for 2h

Read the OD595nm in a micro-titer plate reader

I) BCA Steps


BSA 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0.1 Mean A595 -

Blank 0.

54 0.5 0.46 0.42 0.39 0.31 0.25 0.2 0.13 0.08 0.04

Sample

dilution Conc. 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512

Mean A595 -

Blank 2.18 1.59 0.97 0.52 0.31 0.18 0.11 0.08 0.02 0.01

II) Plotting and calculation of protein concentration


Absorbance (O.D) of standard BSA y = 0.267x + 0.0324

R2 = 0.9898

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1 1.5 2 2.5

BSA concentration (mg/ml)

An

srb

an

ce (

nm

)

Absorbance at 1/8 dilution (0.52 nm) was used to calculate protein concentration in the sample because it is the

first one within range of standard.

Plotting and calculation of protein concentration

y = 0.267x + 0.0324

0.52 = 0.267x + 0.0324

0.4876 = 0.267x

x = 1.826

Concentration at 1/8 is 1.826 mg/ml, then concentration of undiluted protein

equals to

8 (i.e. dilution factor) x 1.826 = 14.608mg/ml

ENZYME LINKED IMMUNOSORBENT

ASSAY (ELISA)

An immunoassay tool used in research and biomedical sciences.

Ag-Ab interaction



Antigen (AP/HRP labeled or unlabeled)

Blocking solution (milk, BSA, Casein etc..), wash solution

Antibodies (I° or II°), may also be labeled (conjugated) or unlabeled

Chromogenic substrate (TMB, AP, OPD, ABTS, etc..)

Spectrophotometer 96 well ELISA plate

Requirements for solid-phase ELISA

Basic ELISA methods



Steps of Indirect & sandwich ELISA


Steps of a direct solid-phase ELISA


Stages in ELISA

TMB or ABTS

catalyzed by HRP enzyme=

product bluish-greenish color

Guide to pipetting



Possible mishaps with coated antibody

WISHING YOU A SUCCESSFUL

PRACTICAL

THANKYOU

&


Documents

GENERAL PRACTICAL (I) INTRODUCTORY LECTUREipmbgazette.weebly.com/uploads/1/0/3/0/1030249/practical_ppt_1.pdf · INTRODUCTION Objectives: General practical (I) preparatory lecture